Journal: Translational Andrology and Urology

Article Title: Functional characterization of the immunomodulatory properties of human urine-derived stem cells

doi: 10.21037/tau-21-506

Figure Lengend Snippet: USCs inhibited the proliferation of PBMNCs in MLR. USC-TA+, USC-TA−, BMSCs, and SMCs were used as stimulator cells and human PBMNCs from two healthy donors were used as responder cells. One-way MLR with single donor PBMNCs (donor A or B) as responder cells and two-way MLR with aliquot half dose of two different donors’ PBMNCs (donor A and B) as responder cells were detected in the same multi-well plate. Cultures were incubated at 37 °C for 5 days in 5% CO2 and 100% humidity, the cells were checked with inverted microscope on the 5th day before further experiment. There were different cell densities of PBMNCs be noticed in different wells for 5 days mix-culture, Scale bar =50 µm (A). The proliferation of PBMNCs was assessed with the BrdU cell proliferation colorimetric ELISA. Newly synthesized BrdU-DNA was quantified using a scanning multi-well spectrophotometer (B). *, P<0.05, post-hoc paired-comparisons. MLR, mixed lymphocyte reaction; BMSC, human bone marrow mesenchymal stem cells; SMC, human smooth muscle cells; USC TA+, human urine derived stem cells with high telomerase activity; USC TA−, human urine derived stem cells with low telomerase activity; PBMNC, human peripheral blood mononuclear cells; SMC + PBMNC A, one-way mixed lymphocyte reaction as human smooth muscle cells mixed with human peripheral blood mononuclear cells from donor A; SMC + PBMNC B, one-way mixed lymphocyte reaction as human smooth muscle cells mixed with human peripheral blood mononuclear cells from donor B; SMC + PBMNC A + B, two-way mixed lymphocyte reaction as human smooth muscle cells mixed with aliquot half dose of two different donors’ human peripheral blood mononuclear cells from donor A and donor B; BMSC + PBMNC A, one-way mixed lymphocyte reaction as human bone marrow mesenchymal stem cells mixed with human peripheral blood mononuclear cells from donor A; BMSC + PBMNC B, one-way mixed lymphocyte reaction as human bone marrow mesenchymal stem cells mixed with human peripheral blood mononuclear cells from donor B; BMSC + PBMNC A + B, two-way mixed lymphocyte reaction as human bone marrow mesenchymal stem cells mixed with aliquot half dose of two different donors’ human peripheral blood mononuclear cells from donor A and donor B; USC + PBMNC A, one-way mixed lymphocyte reaction as human urine derived stem cells mixed with human peripheral blood mononuclear cells from donor A; USC + PBMNC B, one-way mixed lymphocyte reaction as human urine derived stem cells mixed with human peripheral blood mononuclear cells from donor B; USC + PBMNC A + B, two-way mixed lymphocyte reaction as human urine derived stem cells mixed with aliquot half dose of two different donors’ human peripheral blood mononuclear cells from donor A and donor B.

Article Snippet: Human peripheral blood mononuclear cells (PBMNCs, normal human, ATCC PCS-800-011) from two different donors were purchased from ATCC (American Type Culture Collection, MD, USA).

Techniques: Incubation, Inverted Microscopy, Enzyme-linked Immunosorbent Assay, Synthesized, Spectrophotometry, Derivative Assay, Activity Assay

Journal: Translational Andrology and Urology

Article Title: Functional characterization of the immunomodulatory properties of human urine-derived stem cells

doi: 10.21037/tau-21-506

Figure Lengend Snippet: USC showed a characteristic cytokine release profile. The supernatants from USC-TA+, USC-TA−, and BMSCs cultured alone or co-cultured with PBMNCs (direct mixed culture with PBMNCs, or indirect mixed culture with PBMNCs in transwell insert for 48 h) (A) were assessed for their relative levels of cytokines and chemokines by using the human cytokine array panel A, as described in “Methods” (B). A: human cytokine array panel template; B: PBMNCs background; C: USC-TA+ culture alone; D: USC-TA− culture alone; E: BMSCs culture alone; F: USC-TA+ direct mix culture with PBMNCs; G: USC-TA− direct mix culture with PBMNCs; H: BMSCs direct mix culture with PBMNCs; I: USC-TA+ in-direct mix culture with PBMNCs in transwell insert; J: USC-TA− in-direct mix culture with PBMNCs in transwell insert; K: BMSCs in-direct mix culture with PBMNCs in transwell insert: The immunoblot of cytokine expression levels were visualized and quantifed (C). *, P<0.05, comparing with BMSCs alone; †, P<0.05, comparing direct and indirect mixed culture with PBMNCs. BMSC, human bone marrow mesenchymal stem cells; SMC, human smooth muscle cells; USC TA+, human urine derived stem cells with high telomerase activity; USC TA−, human urine derived stem cells with low telomerase activity; PBMNC, human peripheral blood mononuclear cells.

Article Snippet: Human peripheral blood mononuclear cells (PBMNCs, normal human, ATCC PCS-800-011) from two different donors were purchased from ATCC (American Type Culture Collection, MD, USA).

Techniques: Cell Culture, Western Blot, Expressing, Derivative Assay, Activity Assay

Journal: STAR Protocols

Article Title: Mouse splenocyte enrichment strategies via negative selection for broadened single-cell transcriptomics

doi: 10.1016/j.xpro.2022.101402

Figure Lengend Snippet: Flow cytometry enriched antibody cocktail (22 μL) panel for analyzing rarer granulocyte and myeloid cells in mouse spleen for downstream scRNA-seq

Article Snippet: CD11b Antibody, anti-mouse, VioBlue®, REAfinityTM Clone REA592 , Miltenyi Biotec , Cat# 130-113-810; RRID: AB_2726327.

Techniques: Flow Cytometry, Marker

Journal: STAR Protocols

Article Title: Mouse splenocyte enrichment strategies via negative selection for broadened single-cell transcriptomics

doi: 10.1016/j.xpro.2022.101402

Figure Lengend Snippet:

Article Snippet: CD11b Antibody, anti-mouse, VioBlue®, REAfinityTM Clone REA592 , Miltenyi Biotec , Cat# 130-113-810; RRID: AB_2726327.

Techniques: Recombinant, Sterility, Saline, Extraction, Blocking Assay, Software, Solvent, Sequencing, RNA Sequencing, Transferring, Aerosol, Irradiation, Inverted Microscopy, Flow Cytometry, Fluorescence

Journal: Oncotarget

Article Title: Reduction of RKIP expression promotes nasopharyngeal carcinoma invasion and metastasis by activating Stat3 signaling

doi:

Figure Lengend Snippet: A . a representative result of RKIP and phospho-Stat3 immunohistochemical staining in normal nasopharyngeal mucosa (a), NPC without metastasis (b), NPC with metastasis (c), and lymphonode metastasis (d). Original magnification, ×200. B . Kaplan-Meier survival analysis for NPC patients according to the expression levels of RKIP. NPC patients with low RKIP expression have a significantly worse overall survival than those with high RKIP expression. The log-rank test was used to calculate p value.

Article Snippet: Briefly, tissue sections were treated with an antigen retrieval solution [10 mmol/L sodium citrate buffer (pH 6.0)]; incubated with mouse monoclonal anti-RKIP antibody (1:800 dilution) (CST; #13006), or rabbit polyclonal anti-phospho-Stat3(Tyr705) antibody (1: 400) (CST; #9145) overnight at 4°C; and then were incubated with 1:1000 dilution of biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Oncotarget

Article Title: Reduction of RKIP expression promotes nasopharyngeal carcinoma invasion and metastasis by activating Stat3 signaling

doi:

Figure Lengend Snippet: Expression of RKIP and phospho-Stat3 in 127 nasopharygeal carcinomas

Article Snippet: Briefly, tissue sections were treated with an antigen retrieval solution [10 mmol/L sodium citrate buffer (pH 6.0)]; incubated with mouse monoclonal anti-RKIP antibody (1:800 dilution) (CST; #13006), or rabbit polyclonal anti-phospho-Stat3(Tyr705) antibody (1: 400) (CST; #9145) overnight at 4°C; and then were incubated with 1:1000 dilution of biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO).

Techniques: Expressing

Journal: Oncotarget

Article Title: Reduction of RKIP expression promotes nasopharyngeal carcinoma invasion and metastasis by activating Stat3 signaling

doi:

Figure Lengend Snippet: Association between RKIP expression and clinicopathological characteristics in 127 nasopharyngeal carcinomas

Article Snippet: Briefly, tissue sections were treated with an antigen retrieval solution [10 mmol/L sodium citrate buffer (pH 6.0)]; incubated with mouse monoclonal anti-RKIP antibody (1:800 dilution) (CST; #13006), or rabbit polyclonal anti-phospho-Stat3(Tyr705) antibody (1: 400) (CST; #9145) overnight at 4°C; and then were incubated with 1:1000 dilution of biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO).

Techniques: Expressing

Journal: Oncotarget

Article Title: Reduction of RKIP expression promotes nasopharyngeal carcinoma invasion and metastasis by activating Stat3 signaling

doi:

Figure Lengend Snippet: Univariate and multivariate analyses of selected prognostic factors for overall survival using Cox regression model (N=127)

Article Snippet: Briefly, tissue sections were treated with an antigen retrieval solution [10 mmol/L sodium citrate buffer (pH 6.0)]; incubated with mouse monoclonal anti-RKIP antibody (1:800 dilution) (CST; #13006), or rabbit polyclonal anti-phospho-Stat3(Tyr705) antibody (1: 400) (CST; #9145) overnight at 4°C; and then were incubated with 1:1000 dilution of biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO).

Techniques: Expressing

Journal: Oncotarget

Article Title: Reduction of RKIP expression promotes nasopharyngeal carcinoma invasion and metastasis by activating Stat3 signaling

doi:

Figure Lengend Snippet: A . (left) a representative result of Western blotting shows the levels of RKIP expression in 5-8F and 6-10B NPC cells and their transfectants; (right) histogram of relative RKIP expression levels in 5-8F and 6-10B NPC cells and their transfectants as determined by densitometric analysis. B . (left top) a representative result of scratch wound-healing assay shows migration of 6-10B and 5-8F cells and their transfectants. Images were taken at 0, 24 and 48h after wounding under the inverted microscope; (left bottom) a representative result of Matrigel invasion assay shows invasion of 6-10B and 5-8F cells and their transfectants. Invasive cancer cells were photographed at 48h after incubation; (right) histogram of average numbers of invasive cancer cells per microscopic field. C . in vivo metastasis assays of NPC cells with RKIP expression changes. (left) RKIP overexpression 5-8F cells, RKIP knockdown 6-10B cells, and their corresponding empty vector-transfected cells were injected into the tail vein of nude mice, and the representative photography of lung is shown; (middle) histogram of average numbers of lung metastases per mouse; (right) the representative H&E staining of lung tissues shows metastatic tumors. Metastases generated by RKIP overexpression 5-8F cells are significantly less than those generated by empty vector-transfected 5-8F cells. The empty vector-transfected 6-10B cells do not generate metastases, but RKIP knockdown 6-10B cells can generate metastases. Columns, mean values; bars, S.D. *, p < 0.01. Vector, transfcted with an empty vector; OE, overexpression; KD, knockdown.

Article Snippet: Briefly, tissue sections were treated with an antigen retrieval solution [10 mmol/L sodium citrate buffer (pH 6.0)]; incubated with mouse monoclonal anti-RKIP antibody (1:800 dilution) (CST; #13006), or rabbit polyclonal anti-phospho-Stat3(Tyr705) antibody (1: 400) (CST; #9145) overnight at 4°C; and then were incubated with 1:1000 dilution of biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO).

Techniques: Western Blot, Expressing, Wound Healing Assay, Migration, Inverted Microscopy, Invasion Assay, Incubation, In Vivo, Over Expression, Knockdown, Plasmid Preparation, Transfection, Injection, Staining, Generated

Journal: Oncotarget

Article Title: Reduction of RKIP expression promotes nasopharyngeal carcinoma invasion and metastasis by activating Stat3 signaling

doi:

Figure Lengend Snippet: A . mRNA expression levels of E-cadherin, N-cadherin and Vimentin in 5-8F and 6-10B NPC cells and their transfectants detected by qRT-PCR. Columns, mean values from triplicate experiments; bars, S.D. *, p < 0.01. B . a representative result of Western blotting shows the levels of E-cadherin, N-cadherin and Vimentin in 5-8F and 6-10B NPC cells and their transfectants. C . a representative result of immunofluorescent staining shows the levels of E-cadherin, N-cadherin and Vimentin in RKIP overexpression 5-8F cells, RKIP knockdown 6-10B cells and their corresponding empty vector-transfected cells. D . (left) a representative result of immunohistochemical staining of RKIP, E-cadherin, N-cadherin, Vimentin and phospho-Stat3 in the lung metastases of RKIP overexpression 5-8F cells, RKIP knockdown 6-10B cells and empty vector-transfected 5-8F cells; (right) histogram of expression levels of RKIP, E-cadherin, N-cadherin, Vimentin and phospho-Stat3 in the lung metastases. Original magnification, ×200. Columns, mean values from 10 mice; bars, S.D. *, p < 0.01. E-cad, E-cadherin; N-cad, N-cadherin; Vim, Vimentin. Vector, transfcted with an empty vector; OE, overexpression; KD, knockdown.

Article Snippet: Briefly, tissue sections were treated with an antigen retrieval solution [10 mmol/L sodium citrate buffer (pH 6.0)]; incubated with mouse monoclonal anti-RKIP antibody (1:800 dilution) (CST; #13006), or rabbit polyclonal anti-phospho-Stat3(Tyr705) antibody (1: 400) (CST; #9145) overnight at 4°C; and then were incubated with 1:1000 dilution of biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Over Expression, Knockdown, Plasmid Preparation, Transfection, Immunohistochemical staining

Journal: Oncotarget

Article Title: Reduction of RKIP expression promotes nasopharyngeal carcinoma invasion and metastasis by activating Stat3 signaling

doi:

Figure Lengend Snippet: A . a representative result of Western blotting shows the phosphorylated and total levels of Stat3, ERK-1/2, IKK-α/β, IκB-α, and GSK-3β in RKIP overexpression 5-8F cells, RKIP knockdown 6-10B cells and their corresponding empty vector-transfected cells. B . a representative result of immunofluorescent staining shows the nuclear translocation of phospho-Stat3 in RKIP overexpression 5-8F cells, RKIP knockdown 6-10B cells and their corresponding empty vector-transfected cells. C . Stat3 luciferase reporter activity in RKIP overexpression 5-8F cells, RKIP knockdown 6-10B cells and their corresponding empty vector-transfected cells. Columns, mean values from triplicate experiments; bars, S.D. *, p < 0.01. D . a representative result of Co-IP shows RKIP interacting with and inhibiting Stat3 phosphorylation in RKIP overexpression 5-8F cells. Cells were lysed in RIPA buffer, a portion of the sample was removed as the IP input and the remaining supernatant was immunoprecipitated with RKIP antibody and protein A agarose. Immunocomplexes were separated by SDS-PAGE, transferred onto PVDF membrane, and detected with Stat3, phospho-stat3 or RKIP antibody. The input samples were examined for the expression of the indicated proteins. Vector, transfcted with an empty vector; OE, overexpression; KD, knockdown.

Article Snippet: Briefly, tissue sections were treated with an antigen retrieval solution [10 mmol/L sodium citrate buffer (pH 6.0)]; incubated with mouse monoclonal anti-RKIP antibody (1:800 dilution) (CST; #13006), or rabbit polyclonal anti-phospho-Stat3(Tyr705) antibody (1: 400) (CST; #9145) overnight at 4°C; and then were incubated with 1:1000 dilution of biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO).

Techniques: Western Blot, Over Expression, Knockdown, Plasmid Preparation, Transfection, Staining, Translocation Assay, Luciferase, Activity Assay, Co-Immunoprecipitation Assay, Phospho-proteomics, Immunoprecipitation, SDS Page, Membrane, Expressing

Journal: Oncotarget

Article Title: Reduction of RKIP expression promotes nasopharyngeal carcinoma invasion and metastasis by activating Stat3 signaling

doi:

Figure Lengend Snippet: A . a representative result of Western blotting shows the levels of E-cadherin, N-cadherin and Vimentin in RKIP overexpression 5-8F cells transfected with pReceiver-M13-Stat3 or empty vector pReceiver-M13, and RKIP knockdown 6-10B cells treated with a range 0-7.5 μM Stat3 inhibitor Stattic. B . a representative result of scratch wound-healing assay (top) and Matrigel invasion assay (middle) of RKIP overexpression 5-8F cells transfected with pReceiver-M13-Stat3 or pReceiver-M13, and RKIP knockdown 6-10B cells treated with Stat3 inhibitor Stattic; (bottom) histogram of average numbers of invasive cancer cells per microscopic field in these cells. Columns, mean values; bars, S.D. *, p < 0.01. Vector, transfcted with an empty vector; OE, overexpression; KD, knockdown.

Article Snippet: Briefly, tissue sections were treated with an antigen retrieval solution [10 mmol/L sodium citrate buffer (pH 6.0)]; incubated with mouse monoclonal anti-RKIP antibody (1:800 dilution) (CST; #13006), or rabbit polyclonal anti-phospho-Stat3(Tyr705) antibody (1: 400) (CST; #9145) overnight at 4°C; and then were incubated with 1:1000 dilution of biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO).

Techniques: Western Blot, Over Expression, Transfection, Plasmid Preparation, Knockdown, Wound Healing Assay, Invasion Assay